For analytical purposes, the components of a substance mixture can be separated by gas chromatography (GC), liquid chromatography (HPLC), thin-layer chromatography (TLC), capillary electrophoresis (CE), polyacrylamide gel electrophoresis (PAGE), ion mobility spectrometry (IMS) and other, similar methods. It is specific to all these methods that, in each case, only a small amount of the substance mixture is introduced, and that different migration rates occur for the various components of the mixture, leading to a spatial and/or temporal separation of the components. The individual components leave the respective system in the form of small substance batches (“peaks”), or they are available at the end of the separation process in the form of small local accumulations (“spots” or “bands”). So this is by no means a stationary separation process with constant infeed of mixture at one point and constant sampling of a mixture component at another point. Therefore, none of these methods can be adjusted to the sample quantity by varying the duration, and they cannot collect components of low concentration for enrichment.
There are very few separation systems for mixtures which operate as stationary systems. Most of them can be found in big industry, column distillation being one such example. For analytical micro-preparations, hardly any stationary separation systems are known.
Only in the field of ion mobility has a separation system become known which has stationary operation. The system in question is a “High-Field Asymmetric Waveform Ion Mobility Spectrometer” (FAIMS). If a high alternating voltage is interposed between two concentric tubes, an asymmetric alternating field is formed. In this field, ions introduced migrate to one of the two usually tubular electrodes by virtue of the nonlinear components of their mobility. By superimposing a DC voltage it is now possible to create an equilibrium for precisely one ion species so that precisely this ion species is collected in the space between the two tubes. The ions can be introduced into the system at one point as a mixture and sampled at another point as a separated ion species. The disadvantage here is that the separation system is one which can only be operated as an ion filter: one ion species gets through, but all other ion species are destroyed at the electrodes. A further disadvantage is that there is no active transport of the ion species selected from the infeed point to the sampling point.
The ion mobility at an electric field intensity E obeys the simple law v=K×E, where v is the speed of the ion migration. K is a constant which is a function of the friction cross section of the ions and is thus specific to one ion species. K is called the mobility of the ion species. In general, the mobility K is not independent of the field intensity E, however; and the speed v is thus not simply proportional to the field intensity.
On the contrary, the relationship here is:K(E)=K0×(1+K1×E2+K2×E4+ . . . )
Here, K0 is the mobility for vanishingly small electric fields. This dependence of the mobility on the field intensity E means that an ion species subjected to an asymmetric alternating voltage migrates in the direction of the field, even though the temporal integral over the voltage profile of the alternating voltage is exactly zero. An asymmetric alternating voltage in this sense is a voltage which has a high voltage maximum toward one side, toward the positive side, for example, but only for a short time, while toward the other side, here toward the negative side, there is only a low voltage but one which lasts much longer. If the constants K1 and K2 are not zero, this asymmetry brings about a migration in one of the two field directions.
It has not been clarified with certainty why the mobility K is a function of the electric field intensity. One hypothesis is that there are variable states of the solvate envelopes around the individual ions, which are always present even in the gaseous state, said envelopes being able to be more or less skimmed off by collisions with ambient gas or friction with the ambient liquid if the migration rate is high. This then changes the cross section, and hence the mobility. For ion mobility spectrometry, it is known that several water molecules are always to be found on the ions, and that these water molecules are subject to a very rapid and constant interchange.
It could also be another type of conformity change of the ions, however. If the molecule has a dipole in addition to its charge then this dipole can be pulled apart in the field. At a high field intensity, the molecule thus becomes longer and thinner, its cross section changes and thus its mobility in the ambient medium. Further mechanisms for conformity changes are conceivable.
The conformity change does not have to occur immediately, it can also have a settling time. To utilize this conformity change for the separation of substances, however, it is always necessary to let the conformity change occur so that it is also detectable, or even to wait until an equilibrium has been reached. This requirement means there is an upper limit for the frequency of the asymmetric alternating field.
Therefore, it is one object of the invention is to provide a stationary separation system for analytical samples. The separation system will preferably also be able to operate in multichannel mode. The separation system will also be suitable for use especially with protein or peptide mixtures. Many mixtures, including peptides and proteins, contain predominantly charged molecules in aqueous solution; the charge averaged over the molecules of a peptide and over time is dependent on the pH value of the solution. The number of charges of a molecule in solution is not an integer, as is the case with gaseous ions, but is only a time average over a continuously oscillating process of ionization and deionization.